Carbapenemase-producing bacteria isolates (i.e. carbapenemase producers) are increasingly identified throughout the world (1-3). Their early detection is becoming a major issue in the field of clinical microbiology in order to prevent their spread and preserve the efficacy of carbapenems which are becoming the antibiotics of last resort for treating severe infections (4). Indeed, carbapenemases are usually associated to many other non-beta-lactam resistant determinants giving rise to multidrug and pandrug resistance. Moreover, due to current population exchange and travel, early recognition of carbapenemase producers is becoming mandatory whatever the antibiotic policy or rate of multidrug-resistant nosocomial infections.
The vast majority of acquired carbapenemases belong to three of the four known classes of beta-lactamases, namely Ambler class A, Ambler class B (metallo-beta-lactamases (MBLs)) and Ambler class D (oxacillinases (OXAs)). These three classes of carbapenemases confer significant clinical resistance to carbapenems or decreased susceptibiility to carbapenems (1-4). Consequently, carbapenemase-producing bacteria isolates from these three classes have been involved both in nosocomial and community-acquired infections.
The spread of the three distinct classes of carbapenemases varies significantly worldwide. For example, KPC producers (Ambler class A) are identified mostly in the Americas and Southern Europe, while IMP, VIM, NDM-1 (Ambler class B) are extensively identified worldwide with a main reservoir for NDM-1 in the Indian subcontinent. As for OXA-48-like enzymes (Ambler class D) are identified at least in the southern and eastern parts of the Mediterranean coast and more recently in Europe (5).
Currently, there exist two types of methods for detecting carbapenemase producers. First, phenotypic-based techniques for in vivo production of carbapenemase such as the “Etest®” and the “Hodge-Test” can be used (4). The “Etest®”, is a quantitative technique for determining the antimicrobial susceptibility of many microorganisms. The system comprises a predefined antibiotic gradient which is used to determine the Minimum Inhibitory Concentration (MIC), in μg/mL, of different antimicrobial agents against microorganisms as tested on agar media using overnight incubation. As for the “Hodge-Test”, carbapenemase production is detected when the isolate produces the enzyme and allows growth of the carbapenem susceptible strain towards a cabapenem disk. The result of the “Hodge-Test” is a characteristic cloverleaf-like indentation. Unfortunately though, these phenotypic-based techniques are neither sensitive nor specific enough. In many cases also, false-positives have been reported. Alternatively, a molecular detection technique for carbapenemase genes may be used. This technique however remains quite expensive and requires a high degree of expertise. A final drawback of both the phenotypic-based techniques and molecular detection technique is that they are time consuming (12 to 24 h) and therefore do not fulfil clinical requirements requested to implement preventive isolation measures for avoiding development of nosocomial outbreaks (4).
Previous works have performed beta-lactamase identification using a chromogenic cephalosporin such as nitrocefin and CENTA (6, 7). However, these chromogenic substrate molecules cannot specifically detect carbapenemases; they detect any beta-lactamase whatever their hydrolysis profile. As for the Cica-β-test, this test uses the chromogenic cephalosporin HMRZ-86 along with specific inhibitors. Although this test may detect MBL producers, it requires a further culture step. Indeed, the pathogen must first be isolated on an appropriate non-selective medium before being tested. Then, only an isolated colony is used in order to avoid contamination and ensure that the organism is pure. Other tests, such as iodometric tests and acidimetric tests using benzylpenicillin as substrates have been used, but are also not specific for the detection of carbapenemases (6). Finally, techniques using imipenem containing starch agar have also been used to detect MBL activity (8). However, this last technique requires protein extraction, partial beta-lactamase purification, electrophoresis migration as well as an extensive knowledge of the beta-lactamase field. It is therefore time consuming and is usually reserved for research purposes only.
To facilitative the detection of carbapenemases-producers in the field of clinical microbiology, the Applicant has developed a new method based on a simple acido-colorimetric technique. This method is based on the concept that by hydrolysing the beta-lactam ring of a carbapenemase substrate, the carbapenemases generate a carboxyl group which in turn acidifies a medium. The acidity resulting from this hydrolysis is then identified by a color change of a pH color indicator (9).
This method helps differentiate the carbapenemase producers from those that are carbapenem resistant resulting from non-carbapenemase mediated mechanisms such as combined mechanisms of resistance (eg. outer membrane permeability defect, overproduction of cephalosporinases, clavulanic-acid inhibited ESBL . . . ) or from strains expressing broad-spectrum β-lactamases without carbapenemase activity (ESBLs, plasmid and chromosome-encoded cephalosporinases) (10).
Interpretable results are obtained within a very short time, which is crucial when designing containment measures for carbapenemase producers. It eliminates the need of using the “Etest®” or “Hodge-Test” technique which, as mentioned previously, is neither specific nor sensitive and which needs an additional 18 h before obtaining interpretable results.
This method offers a solution for fast, reliable and affordable detection of any type of carbapenemases-producers. In addition, it is specific and sensitive. It may also be submitted to an industrialization process such that it may be implemented in any clinical microbiology laboratory worldwide without significant additional workload for laboratory technicians.
Moreover, in the field of epidemiology, the use of this method may be of further help when wanting to rapidly select strains which should be tested by PCR and sequenced for identification of carbapenemase genes.